An experimental system is proposed for studying viral evolution in response to major changes in its genome: deletions, gene rearrangements, gene additions, and wholesale changes in regulation. Many of these genome changes will cause major drops in viral fitness. Can these modified viruses evolve back to high fitness, just as viruses have been extremely versatile in evolving resistance to drugs? The work will use the bacteriophage T7, which is amenable to a wide scope of genomic alterations and for which the supporting molecular genetics and biochemistry is extensive enough to develop a predictive framework for evolution and to interpret the evolutionary mechanisms. The three specific aims are: 1) Characterize regulatory evolution in viruses with modified, complete genomes; 2) Determine whether viruses with engineered deletions can evolve to recover original fitness levels; 3) Observe the evolution of viral genomes with new genes. Genome alterations will be performed, the altered viruses will be grown extensively to allow improvement through evolution, and the evolved genomes will be analyzed by sequencing, mapping beneficial changes, and site-directed mutation to test the effects of different mutations. The combined studies will yield an understanding of the mechanisms by which viral genomes evolve and maintain high fitness across a variety of genome modifications. The work developed here will have relevance to the development of live, attenuated vaccines, the improvement of phages as antibacterial agents (phage therapy), and to assess the long-term consequences of any viruses engineered to perform specific functions.